The Neddylation pathway was recently validated as a cancer target. The SENP8 protease processes the precursor of Nedd8 and is essential for its activation. Based on the high resolution crystal structure of Nedd8 bound to SENP8 the substrate has two fundamental interaction sites: at the C-terminus of Nedd8 in the catalytic center, and interactions with the bulk of the Nedd8 protein covering a substantial surface of the protease - the exosite. Analysis of the SENP8 structure reveals cavities in the exosite, indicating the presence of a putative allosteric binding site. Our preliminary results for this SENP on uHTS of the ~ 330,500 MLSMR small molecule libraries using a penta-peptide based assay identified inhibitors binding to the catalytic center and not selective against other SENPs. In order to find selective SENP8 ligands and to explore this additional allosteric binding site, we developed a novel Fluorescent Intensity uHTS assay that utilizes a physiological protein substrate Nedd8 substrate. To study the function of this therapeutically important enzyme, we propose to screen for selective mechanistically novel SENP8 inhibitors using this newly developed assay. Based on available literature, this would be the first time that a representative protease targeting ubiquitin-like proteins is interrogated using a full-length substrate following an uHTS screen that targets only the catalytic site. Our application addresses an important unmet need for identification of specific modulators of SENPs. We will utilize the compounds identified in the project for characterization of physiological involvement of this class of enzymes in maintaining homeostasis of normal cells and their role in cancer. These compounds will be made available to other research labs, permitting acceleration of research in the SENP field. PUBLIC HEALTH RELEVANCE: Nedd8 is an ubiquitin-like protein, and its attachment to target proteins, by a process known as neddylation, is essential for specific cellular pathways. Previous work has shown that targeting the neddylation cycle with a small molecule can decrease tumor burdens, and this small molecule is now in clinical trials. To study the function of this therapeutically important enzyme, we propose to deploy a novel Fluorescent Intensity uHTS assay that utilizes a full length Nedd8 protein substrate to discover potent and SENP8 selective inhibitors that may prove to be allosteric using this newly developed assay.